This invention relates to a process for conducting a two-step sample treatment process in a gel which involves a step of transferring a shaped gel cylinder containing samples from a first step of treatment to a second sample separation step.
Electrophoresis is the resolution of a complex mixture of macromolecules on the basis of charge and/or size under the influence of an electric field and is a primary tool in analytic chemistry, used to separate complex mixtures of molecules such as proteins into their individual components. Electrophoretic analysis is based upon the fact that each molecule is characterized by a particular electrophoretic mobility under a given set of conditions. Macromolecules will migrate within a voltage gradient according to their net charge and will reach equilibriun at their isoelectric point at which their net mobility will be zero. For example, many proteins exhibit a net negative charge which is affected by the surrounding pH. When a mixture of proteins is placed in a support medium, such as a buffered gel, which is subjected to a voltage gradient, each component is caused to migrate through the support medium at its characteristic rate for that set of conditions. Electrophoretic mobility is a function of net charge, molecular weight, shape and a number of other factors which are controlled by experimental conditions.
Two dimensional gel electrophoresis permits resolution of complex proteins on the basis of charge in one step and on the basis of molecular size in a second step. Either size separation of charge separation can be conducted in the first step. As part of the first step, the sample is added to a shaped gel cylinder which has been cast in a narrow tube, e.g, 3 mm or less diameter or maximum cross sectional dimension, which gel is characterized by a pH gradient. Generally, the top of the gel cylinder has a basic pH of about 10-13 and the bottom of the gel cylinder has an acidic pH of about 2-5 or other pH ranges could be used. A pH gradient is established over the length of the gel. The sample is placed on the gel which then is subjected to a voltage gradient which, in turn, causes the constituents of the sample to migrate through the gel to the point at which the constituent is electrically neutral, i.e., the isoelectric point.
After making the isoelectric focussing separation, the gel is carefully extruded from the tube into an equilibration or storage buffer, and then transferred onto the edge of a second separation medium such as a membrane or a gel slab for the second dimension separation. In the second dimension separation, the sample constituents are separated on the basis of molecular size, charge etc. Numerous methods have been employed to effect this transfer, but it is an extremely difficult laboratory procedure. This is in large part due to the extraordinary fragility of the first dimension gel which cannot support its own weight in air and must be handled under a liquid or on a solid surface to maintain integrity. Typically the gel is pressure extruded from the tube as a glass tube into a buffer solution, care being taken to keep the end of the tube entirely submerged in the buffer during extrusion. The top of the second separation medium to which the shaped gel is to be applied is submerged under an electrophoresis buffer, and the transferred gel cylinder is manipulated commonly under liquid onto the edge of the second gel by careful manipulation with probes and forceps.
Even when handled under liquid, the gel can be readily stretched if insufficient care is not taken thus causing distortion of the resultant final electrophoretic pattern, or even broken while attempting to maneuver the cylinder onto the slab gel. Furthermore, the first dimension gel will tenaciously adhere to a dry surface, if inadvertently contacted during a transfer operation. Therefore, these cylindrical gels often are transferred from their original tube to the slab gel entirely under liquid. This poses an additional problem. The gel is completely transparent and has almost the same refractive index as the buffer solutions used. As a consequence it is extremely difficult to even see the gel during transfer operations, in addition to the mechanical handling problems.
Accordingly, it would be highly desirable to provide a means that allows easy mechanical transfer without distortion and breakage of the gel cylinder. Furthermore, such means should reduce the extraordinary dexterity skills presently required to effect transfer of the gel cylinder.